Method of Peptide Library Construction and Peptides Thereof

Abstract

A method of library construction for peptide generation and, more particularly, to a method of construction of a DNA library built for selection of peptides that starts with a protein sequence known to bind to a target, wherein the protein sequence is used to generate DNA sequences which are then recombined and wherein each codon of the recombined DNA sequences has degenerate bases that cover between 1 and 20 of 20 possible amino acids.

What is claimed is: 
     
         1 . A method of constructing a peptide library, comprising:
 1) identifying a protein of interest;   2) synthesizing DNA primers comprising DNA sequences, wherein the DNA sequences are determined by the following steps:
 a) writing a DNA sequence that codes for the identified protein or a segment thereof; 
 b) splitting the written DNA sequence of the identified protein or segment thereof into a plurality of written DNA sequences from 3 to 18 nucleotides or longer, thereby forming a list of short written DNA sequences; 
 c) introducing degeneracy into one or more of the short written DNA sequence from step (b) to create one or more short degenerate written DNA sequences; and 
 d) adding the one or more short degenerate written DNA sequences to written DNA primer sequences designed to amplify a DNA fragment of a screening construct to create written DNA primer sequences with overhangs; and 
 e) synthesizing the written DNA primer sequences with overhangs from step (d) to create synthesized primers; 
   3) amplifying DNA fragments using at least three or more of the synthesized primers from step (e) and one or more DNA polymerases;   4) mixing, phosphorylating and ligating the amplified DNA fragments from step (3), thereby forming a DNA library; and   5) expressing peptides coded by the DNA library formed in step (4) in an appropriate system in vitro or in vivo, thereby forming a first peptide library having peptides with a pharmacologic activity.   
     
     
         2 . The method of  claim 1 , wherein in step (c), degeneracy is introduced into the one or more short written DNA sequences by one of the following steps:
 changing the nucleotide at the first position of every codon;   changing the nucleotide at the second position of every codon;   changing the nucleotide at the first position of the first codon, the second position of the second codon and repeating this pattern for the entire short written DNA sequence;   changing the second position of the first codon and the first position of the second codon and repeating this pattern for the entire short written DNA sequence; and   changing the first position and/or the second position of less than every codon.   
     
     
         3 . The method of  claim 1 , wherein the segment of the identified protein is a binding domain. 
     
     
         4 . The method of  claim 1 , wherein steps (2) through (5) are repeated, thereby forming a second peptide library with an improved pharmacologic activity, compared to the first peptide library. 
     
     
         5 . The method of  claim 1 , wherein the segment of the identified protein consists of loops and/or secondary protein structures. 
     
     
         6 . The method of  claim 3 , wherein the size of the binding domain is at least 220 or more amino acids. 
     
     
         7 . The method of  claim 1 , wherein in step 3, the primers are used simultaneously. 
     
     
         8 . The method of  claim 1 , wherein in step 3, one or more of the synthesized primers are designed for a segment of the binding domain, wherein the segment comprises 10 or more amino acids. 
     
     
         9 . The method of  claim 1 , wherein in step 3, all of the synthesized primers are designed for a segment of the binding domain, wherein the segment comprises 10 or more amino acids. 
     
     
         10 . The method of  claim 1 , wherein amplified DNA fragments from step 3 are digested by type IIS restriction enzymes. 
     
     
         11 . The method of  claim 1 , wherein ligated DNA fragments from step 4 are amplified with the flanking primers. 
     
     
         12 . The method of  claim 1 , wherein each DNA sequence in step (c) is different from the DNA sequence from step (b) by only one codon. 
     
     
         13 . The method of  claim 1 , wherein the degenerate written short DNA sequences in step 3 are linked as overhangs to primers with one forward primer and one reverse primer. 
     
     
         14 . The method of  claim 1 , wherein the one or more DNA polymerases in step (e) comprise a Vent polymerase generating blunt DNA ends. 
     
     
         15 . The method of  claim 1 , further comprising the following steps:
 6) selecting and synthesizing peptides from the first peptide library;   7) measuring an affinity of binding of the selected and synthesized peptides from the first library to the desired biological target; and   8) measuring efficacy of the selected and synthesized peptides from the first library in vitro and/or in vivo.   
     
     
         16 . The method of  claim 4 , further comprising the following steps:
 6) selecting and synthesizing peptides from the second peptide library;   7) measuring an affinity of binding of the selected and synthesized peptides from the second library to the desired biological target; and   8) measuring efficacy of the selected and synthesized peptides from the second library in vitro and/or in vivo.   
     
     
         17 . The method of  claim 16 , wherein the desired biological target is interleukin-6. 
     
     
         18 . A peptide from a peptide library produced by the method of  claim 16 , the peptide comprising the amino acid sequence as set forth in any one of SEQ ID Nos: 54, 55, 70, 73, and 83, or an amido acid sequence having more than 70% homology thereto. 
     
     
         19 . The peptide of  claim 18 , wherein the amino acid sequence has more than 75% homology to any one of SEQ ID Nos: 34, 35, 54, 55, 70, 73, and 83. 
     
     
         20 . The peptide of  claim 18 , wherein the amino acid sequence has more than 80% homology to any one of SEQ ID Nos: 34, 35 54, 55, 70, 73, and 83. 
     
     
         21 . The peptide of  claim 18 , wherein the amino acid sequence has more than 85% homology to any one of SEQ ID Nos: 34, 35, 54, 55, 70, 73, and 83. 
     
     
         22 . The peptide of  claim 18 , wherein the amino acid sequence has more than 90% homology to any one of SEQ ID Nos: 34, 35, 54, 55, 70, 73, and 83. 
     
     
         23 . The peptide of  claim 18 , wherein the amino acid sequence has more than 95% homology to any one of SEQ ID Nos: 34, 35, 54, 55, 70, 73, and 83. 
     
     
         24 . The peptide of  claim 18 , wherein the peptide is SEQ ID No: 55 or an amido acid sequence having more than 70% homology thereto. 
     
     
         25 . The peptide of  claim 24 , wherein the amino acid sequence has more than 75% homology thereto. 
     
     
         26 . The peptide of  claim 24 , wherein the amino acid sequence has more than 80% homology thereto. 
     
     
         27 . The peptide of  claim 24 , wherein the amino acid sequence has more than 85% homology thereto. 
     
     
         28 . The peptide of  claim 24 , wherein the amino acid sequence has more than 90% homology thereto. 
     
     
         29 . The peptide of  claim 24 , wherein the amino acid sequence has more than 95% homology thereto. 
     
     
         30 . The peptide of  claim 18 , wherein the peptide's termini are protected. 
     
     
         31 . The peptide of  claim 18 , wherein the N terminus of the peptide is acetylated and the C terminus is amidated. 
     
     
         32 . A peptide which exhibits antagonistic activity directed against interleukin-6, the peptide comprising the amino acid sequence as set forth in any one of SEQ ID Nos: 34, 35, 54, 55, 70, 73, and 83, or an amido acid sequence having more than 70% homology thereto. 
     
     
         33 . A method of inhibiting IL-6 signaling in a cell, the method comprising contacting a cell with an effective amount of the peptide according to  claim 18 . 
     
     
         34 . A method of treating a disease or condition mediated by IL-6 mechanism comprising administering to a patient in need thereof an effective amount of the peptide according to  claim 18 . 
     
     
         35 . The method of  claim 34 , wherein the disease or condition comprises an inflammatory, degenerative or an autoimmune disease or condition. 
     
     
         36 . The method of  claim 34 , wherein the disease or condition is selected from the group consisting of rheumatoid arthritis, Crohn's disease, Castleman disease, systemic lupus erythematosus, juvenile idiopathic arthritis, giant cell arteritis, ulcerative colitis, psoriatic arthritis, ankylosing spondylitis, multiple myeloma, systemic sclerosis, Still's disease, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19), chronic obstructive pulmonary disease, atherosclerosis, osteoporosis, type 2 diabetes mellitus, depression, Alzheimer's disease, and cytokine release syndrome.